The p53-related protein, Np631, is a potent oncogene driving the development and progression of squamous cell carcinomas (SCC) of various origins. While physiologic levels of this factor are required for epithelial stem cell maintenance, Np631 overexpression promotes cancer cel proliferation and survival, and inhibits cel cycle arrest and apoptosis. The overall goal of this project is to define the mechanisms by which Np631 impacts gene transcription and cell behavior. Our initial findings indicate that in H226 SCC cells, Np631 is a potent gene-specific repressor of the pro-apoptotic factor NOXA, by mechanisms independent of its ability to bind chromatin at p53 responsive elements, and at regulatory steps that follow p53-mediated histone acetylation. Yet in different SCC cell lines, other investigators have shown that Np631 depletion de-represses other p53/p73-responsive genes. The first Specific Aim of this project will define the relative contributions of the other family members, p53 and p73, to the transcriptional and celular efects of Np631 depletion in H226 cells and other cell lines. A panel of isogenic H226 cell lines lacking p53 and/or p73 will be created using stably-expressed shRNA constructs, and the effect of Np631 depletion on cel survival, proliferation, apoptosis, and the expression of >30 p53/p73 target genes will be determined. Furthermore, NOXA and Np631 will be concomitantly knocked down in H226 cells to determine whether NOXA upregulation is crucial for the loss of cell proliferation upon Np631 knockdown. These studies will enable us to define the epistatic relationship between p53/p73, Np631, and NOXA. The second Specific Aim will identify the molecular mechanism by which Np631 represses the expression of specific p53/p73 target genes. By performing an exhaustive chromatin immunoprecipitation (ChIP) analysis of the transcriptional events taking place at the NOXA and p21 loci before and after Np631 knockdown under conditions of both basal and activated p53, the precise regulatory steps affected by Np631 will be identified. The final Specific Aim will determine the impact of biologically-relevant Np631 mutations on gene-specific repression. An in vivo structure-function analysis will be performed by rescuing Np631 knockdown cells with either wild type Np631 or constructs containing naturally occurring mutations in the DNA binding domain, SAM domain, and other regions. These experiments will identify crucial functional regions of Np631 and will further illuminate the Np631 mechanism of action. It is anticipated that the knowledge generated during this project will vastly improve our understanding of Np631, its mechanism of action, and its role in normal epithelial maintenance and oncogenesis, as well as to help enable strategies for the therapeutic control of the various cancers driven by Np631 overexpression.